Project Summary/Abstract The objectives of the research described in this Pathway to Independence Award application are to provide the candidate with training and experience in protein engineering to develop artificial metalloenzymes that catalyze new or improved synthetic reactions to prepare biologically active compounds. The candidate, Levi Stanley, Ph. D., is currently an NRSA/NIGMS postdoctoral fellow with the immediate goal of completing an additional one to two years of mentored research to establish research skills in the field of protein engineering and to apply these new skills to the development of artificial metalloenzymes that combine the broad reactivity of transition metal complexes with the ability of enzymes to exquisitely control the selectivity of synthetic transformations. Dr. Stanley's long-term career goals include establishing himself as an independent scientist in a tenure-track academic position and contributing to the improvement of human health through the development of catalytic reactions that generate bioactive compounds and building blocks to such compounds. Dr. Stanley has established expertise in transition metal catalysis through his current research in Professor John Hartwig's laboratory at the University of Illinois. The career development plan and research strategy will allow the candidate to combine his knowledge of transition metal catalysis with current methods for protein engineering. The mentored phase of the program will be carried out in collaboration with Professor John Hartwig, a leader in the field of transition metal catalysis, and Professor Huimin Zhao, an expert in the field of protein engineering. The established laboratories of the candidate's co-mentors at the University of Illinois provide an ideal environment to conduct research at the interface of their respective areas of expertise. The proposed research in the mentored phase will allow the candidate to create a novel platform for the discovery and development of new artificial metalloenzymes that catalyze reactions that are useful to synthetic chemists. The independent phase of the proposed research will focus on the application of these new artificial metalloenzymes as catalysts in regioselective, chemoselective, and stereoselective C-H functionalization, cyclopropanation, and aziridination reactions. The proposed research program is outlined in four specific aims. The first aim is focused on the identification of metal-substituted carbonic anhydrases that exhibit activity as catalysts for C-H functionalization reactions. The second aim is directed toward developing methods for the generation, expression, purification, and substitution of the metal in mutant carbonic anhydrase libraries. The third aim is designed to improve by directed evolution the activity of metal-substituted carbonic anhydrases that are active for C-H functionalization reactions. The first two aims and the beginning of the third aim will be the primary focus of the mentored (K99) phase and will provide the background necessary for the candidate to conduct protein engineering and directed evolution during the independent phase of the program. The fourth aim is directed toward the divergent evolution of metal-substituted carbonic anhydrases to identify mutants that lead to different regioselectivities and chemoselectivities from a common starting point. Research toward the completion of aims three and four will be conducted during the independent (R00) phase of this program. The results of these studies will establish a new platform to generate and improve artificial metalloenzymes and will lay the groundwork to expand the breadth of transformations catalyzed by metalloenzymes that are useful to synthetic chemists.